210Protective Effect of Aloe vera Gel against Cisplatin-Induced Testicular Damage, Sperm Alteration and Oxidative Stress in Rats

Background: Cisplatin (CIS) is an effective antineoplastic drug that is used to treat various types of cancers. However, it causes side effects on the male reproductive system. The present study aimed to investigate the possible protective effects of Aloe vera (AL) gel (known as an antioxidant plant) on CIS-induced changes in rat sperm parameters, testicular structure, and oxidative stress markers. Materials and Methods: In this experimental study, forty-eight adult male rats were divided into 6 groups including: control, CIS, AL, metformin (MET), CIS+AL, and CIS+MET. CIS was used intraperitoneally at a dose of 5 mg/kg on days 7, 14, 21, and 28 of the experiment. AL gel (400 mg/kg per day) and MET (200 mg/kg per day) were administered orally for 35 days (started one week before the beginning of the experiment). Testes weight and dimensions, and morphometrical and histological alterations, activities of antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GPx), serum testosterone concentration, lipid peroxidation level, and sperm parameters were examined. Results: CIS caused a significant decrease (P<0.05) in relative weight and dimension of the testis, germinal epithelium thickness and diameter of seminiferous tubules, the numbers of testicular cells, and spermatogenesis indexes. The malondialdehyde (MDA) levels increased and antioxidant enzymes activities decreased in the CIS group compared to the control group (P<0.05). Additionally, sperm parameters (concentration, viability, motility, and normal morphology), and testosterone levels reduced significantly in CIS-treated rats (P<0.05). Also, CIS induced histopathological damages including disorganization, desquamation, atrophy, and vacuolation in the testis. However, administration of AL gel to CIS-treated rats attenuated the CIS-induced alterations, mitigated testicular oxidative stress and increased testosterone concentration. Conclusion: The results suggest that AL as a potential antioxidant plant and due to free radicals scavenging activities, has a protective effect against CIS-induced testicular alterations.


Introduction
Cisplatin [CIS-diaminedichloroplatinum (II)] is one of the mos t effective anticancer drugs which is used for treatment of a vas t variety of human cancers. The anticancer activity of CIS is due to multiple mechanisms such as induction of DNA damage, oxidative s tress, and programmed cell death (apoptosis) (1).
Despite the fact that CIS is a useful anticancer drug, it is very toxic and induces several side effects including reproductive toxicity, hepatotoxicity and nephrotoxicity (1,2). Reproductive toxicity is one of the mos t common side effects of CIS in treated patients (2)(3)(4)(5). CIS causes severe tes ticular damage which is characterized by apoptosis of germ cell, dysfunction of Leydig cell, tes ticular s teroidogenic disorder and spermatogenic damage (3)(4)(5)(6). The precise mechanism of reproductive toxicity induced by CIS is not fully es tablished, however oxidative s tress has been known as the major cause of CIS-related tes ticular dysfunction (5,6). Hence, several inves tigators have used antioxidant compounds to reduce reproductive damages caused by CIS (2,5,6). For example, olive leaf extract which contains flavonoid and polyphenolic compounds ameliorated CIS-induced tes ticular oxidative s tress in rats (5). Also, fenugreek seed extract reduced oxidative s tress and tes ticular tissue damage induced by CIS and improved spermatogenesis in the rats (6).
Aloe barbadensis Miller or AL, is a perennial shrubby plant of the Liliaceae family. It considered an important medicinal herb because of its many medicinal activities including antitumor, antioxidant, anti-allergic, anti-viral, and anti-inflammatory properties. It has been proposed that the antioxidant activity of AL may be a major property of this plant used in the treatment of several diseases. The antioxidant property of AL is due to a large amount of antioxidants subs tances such as vitamins (A, C, B and E), flavonoids, phenolic compounds, and polysaccharides (7). Several researchers have provided experimental evidence for the reproprotective effect of AL in experimental animals (8)(9)(10)(11). These s tudies have shown that AL can efficiently attenuate the tes ticular alteration induced by some drugs and heavy metals (8,9). Other s tudies reported that AL due to its antioxidant compounds (especially vitamin E) can improve tes ticular weight, height of the germinal epithelium and diameter of seminiferous tubule, and ameliorate reductions in the number of tes ticular cells. Also, phenolic and flavonoids contents of AL can be effective in increasing the antioxidant enzymes activity and decreasing lipid peroxidation that can cause extensive damage to cell membranes lipids (8)(9)(10)(11).
It has been found that oxidative s tress plays a major role in the pathogenesis of reproductive toxicity induced by CIS. Because of the antioxidant property of AL gel, it was hypothesized that AL may attenuate CIS-mediated gonadotoxicity in rats. Therefore, this s tudy was designed for the firs t time, to examine possible protective effects of AL gel on gonadotoxicity induced by CIS via evaluation of epididymal sperm parameters, alterations of tes ticular tissue, tes tos terone levels, and oxidative/antioxidant markers in the tes tis of rats.

Preparation of A. vera gel and analysis of its antioxidant properties
AL gel powder (A. barbadensis) was obtained from Barij Essence Pharmaceutical Co (Kashan, Iran). Total flavonoids content (TFC) was measured by aluminum chloride colorimetric assay (12). The catechin solutions (0-25 μg/mL) were prepared for flavonoid assessment. Aliquots (25 μL) of each AL gel (10 mg in 1 ml dis tilled water) and s tandard were mixed with 125 μL dis tilled water followed by adding 8 μL of 5% sodium nitrate. After 5 minutes, 0.15 ml of 10% aluminum chloride solution was added to 15 μL of that mixture. The absorbance was measured at 517 nm. TFC is expressed as the percentage of catechin equivalents (QE) per 100 g dry weight, and was determined from the s tandard calibration curve.
Total phenolic content (TPC) of AL gel was es timated using the Folin-Ciocalteu (FC) and aluminum chloride colorimetric assay as described by Im et al. (12). Contents are expressed as the percentage of gallic acid equivalents (GAE) per 100 g dry weight of AL gel.

Animals and experimental groups
In this experimental s tudy, a total of forty-eight healthy male Wis tar rats (180-200 g) were maintained under s tandard laboratory conditions (12-hour light: 12-hour dark at 22 ± 2 °C) and fed with commercial rat pellets (Pars Animal Feed Co, Tehran, Iran) and water. All experimental assays were approved by the Ethics Committee of Shahid Chamran University of Ahvaz for animal and human experiments (EE/99.3.02.15058/ssu.ac.ir).
After a quarantine period of 7 days, the rats were divided randomly into 6 groups (n=8) as follows: control group: rats fed with a s tandard diet and kept in normal conditions. CIS group (CIS): rats received CIS intraperitoneally (i.p) at a dose of 5 mg/kg on days 7, 14, 21, and 28 of the experiment. AL group (AL): AL gel powder was dissolved in dis tilled water and adminis tered orally at a dose of 400 mg/kg/day for 35 days. MET group (MET): rats received MET (200 mg/kg/day, orally) for 35 days. The experiment las ted for 35 days (13). The dose of CIS was selected based on a published report (14). The dosing regimen for AL and MET were selected based on reports by Behmanesh et al. (13) and Sahu et al. (15), respectively.
The blood samples were collected via cardiac puncture and centrifuged (at 3000 rpm for 10 minutes). Serum samples were separated and then s tored at -20˚C for tes tos terone hormone analysis. Afterward, tes tes and epididymis were obtained from the abdominal cavity. The weight, dimensions (length and diameter) and volume of tes tes were measured using a digital scale, a caliper, and water displacement method, respectively (17). The left tes tis was fixed in a 10% buffered formalin solution for his tological analyses and the right tes tes were s tored at -20°C for oxidant/antioxidant assessment. The epididymis tissue samples were used for the analysis of sperm parameters.

The tes ticular index
The relative tes tis weight ratio (%) was calculated using the formula: (absolute weight of the tes tis/ total body weight)×100 (5).

Morphometrical analyses
For this purpose, 100 cross sections of seminiferous were chosen randomly in 5 non-serial sections per animal (10 tubules in the central zone and 10 tubules in the peripheral (sub-capsular) zone of each section). Then, the seminiferous tubule diameter and height of germinal epithelium were measured at ×10 magnification. Also, Sertoli, Leydig, spermatogonia, primary spermatocyte, early spermatid and late spermatid cells were counted in a marked scale (150 µm) at ×40 magnification (18,19). All measurements were performed under a light microscope (Olympus Optical Co., Japan) using Dino-Lite digital lens (with Dino capture software, FDP2, Taiwan).
Spermiogenesis index (SI) and tubular differentiation index (TDI) were calculated for spermatogenesis assay. SI index was calculated using the following formula: (Seminiferous tubules contained sperm/seminiferous tubules without sperm)×100.
For TDI index, the percentage of tubules that contained three or more differentiated spermatogenic cells from the type A spermatogonia (i.e. intermediate or type B spermatogonia, spermatocytes, or spermatids) were calculated (18).

Analysis of sperm parameters
The cauda epididymis was minced finely in (5 ml) Ham's F-10 medium and placed at 37°C for 15 minutes. Spermatozoa in the epididymis were counted by a s tandard hemocytometric method and motility of sperm (progressive, non-progressive, and immotile) was evaluated under a light microscope (Olympus Optical Co., Japan) at 3 consecutive es timates and reported as mean (20). Sperm viability and morphology were evaluated by the methods described by Turk et al. (21) and Adibmoradi et al. (18). Briefly, a 10-μL sperm suspension was slowly mixed with 40 μL eosinnigrosin (1.67% eosin, 10% nigrosin and 0.1 M sodium citrate). Then, 10 μL of this mixture was transferred to a glass slide and spread slowly by another slide. After preparation of smears, viability and morphology of sperms were evaluated. Spermatozoa with red head were classified as dead sperm and spermatozoa with white head were classified as live sperm (18). Also, sperms were screened and classified into normal and abnormal types, and then the percentage of abnormality was determined for each group (21).

Tissue preparation for oxidant/antioxidant markers assay
Here, 100 mg of the right tes ticular tissue sample was homogenized in 500 μL RIPA lysis buffer (1 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 10 mM Tris-HCL; pH=8, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride) by a glass homogenizer (Heidolph, Germany). Homogenate was centrifuged at 10000 rpm for 15 minutes at 4°C (Centrifuge 5415 R; Eppendorf AG, Germany) and the supernatant was collected and s tored at -70°C for subsequent analysis. The protein concentration of the supernatant was es timated using the Bradford method (22).

Analyses of lipid peroxidation levels and antioxidant enzymes activities
The content of malondialdehyde (MDA) in the tes tis was assessed as a lipid peroxidation marker using the thiobarbituric acid reactive subs tance (TBARS) assay with slight modifications (23). The MDA concentration was obtained based on MDA-TBARS complex optical density at 532 nm wavelength in comparison with the s tandard curve of MDA. The MDA results are expressed as nmol/mg of protein. Superoxide dismutase (SOD) activity was determined by the nitro blue tetrazolium (NBT) reduction assay, as described by Kakkar et al. (24). Finally, glutathione-peroxidase (GPx) activity was evaluated by a GPx detection kit according to the manufacturer's ins tructions (RANSEL, Randox Com, UK). Both SOD and GPx activities are expressed as units/mg protein.

Tes tos terone analysis
Tes tos terone concentration in the serum samples of the experimental groups was quantitatively assessed through enzyme-linked immunosorbent assay (ELISA) using the Diametra tes tos terone ELISA kit (Diametra Co, Italy), according to the manufacturer's protocol. Tes tos terone results are expressed as ng/dl.

S tatis tical analysis
Data are expressed as mean ± s tandard deviation and were analyzed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Differences among various groups were assessed by one-way analysis of variance (ANOVA) followed by the Tukey tes t. In all cases, P<0.05 was regarded as significant.

Phytochemical content of A. vera gel
The results showed that concentrations of the total phenol and flavonoid contents in the AL gel were 49.81 μg GAE/mg and 56.42 μg QE /mg of gel powder, respectively.

Relative weight and dimensions of the tes tis
The results showed that CIS caused a significant (P<0.05) decrease in relative weight, length, diameter and volume of both the right and left tes tes compared to the control group. The co-adminis tration of AL and CIS significantly increased relative weight, length and diameter of the tes tes (right and left), and volume of the right tes tis compared to the CIS group (P<0.05). Although, there was a numerical increase in the volume of the left tes tis in the CIS-AL group, it was not s tatis tically significant. Treatment of CIS-treated rats with MET significantly attenuated the reduction of relative weight, length and volume of both the right and left tes tes and diameter of the left tes tis (P<0.05). Also, MET increased the diameter of the right tes tis, however this change was not significant compared to the CIS group (Table 1).

His tological findings
The tes ticular tissue of the control group composed of a high density of normal shape tes ticular tubules surrounded by inters titial connective tissues. Seminiferous tubules lined by a s tratified germinal epithelium, showed features of active spermatogenesis. Spermatogonia cells with heterochromatin and rounded nuclei res ted on the basal lamina. Primary spermatocytes were the larges t spermatogenic cells in the germinal epithelium with different shapes of chromatin. Furthermore, early-s tage spermatids with euchromatin and round nuclei and late-s tage spermatids with heterochromatin and elongated nuclei, were attached to the membrane of Sertoli cells. Also, Sertoli cells res ted on the basal lamina and had large, euchromatin nuclei with prominent nucleolus. The Leydig cells in inters titial connective tissues had eosinophilic cytoplasm with large and round nuclei (Fig.1A). In AL (Fig.1B, AL) and MET (Fig.1C, MET) treated groups, the seminiferous tubules showed normal cells associations without any s tructural changes compared to the control group (Fig.1B,  C). CIS caused atypical morphological features such as disorganization, and desquamation in the seminiferous tubules. Also, widespread atrophy and loss of all germ cells and extensive vacuolation in the epithelium were observed in CIS-treated rats. In addition, maturation arres t and absence of spermatozoa in the lumen in a majority of seminiferous tubules were significant (Fig.1D). Co-adminis tration of AL and CIS normalized these his tological changes and amended spermatogenesis when compared with the CIS alone group; though a slight vacuolation was found, desquamation was s till observed in the seminiferous tubules (Fig.1E). Likewise, MET attenuated the histological abnormalities induced by CIS, and protected the tes ticular tubules although it was less than that seen for AL (Fig.1F).

Morphometrical finding
The number of Sertoli, Leydig, spermatogonia, primary spermatocytes, early and late spermatids cells (Table 2), germinal epithelium thickness, diameter of seminiferous tubule and the spermatogenesis indexes (TDI and SI) decreased in the central and peripheral (sub-capsular) zones of the tes tis after the CIS treatment (P<0.05, Fig.2). But, adminis tration of AL and MET along with CIS significantly res tored these alterations (P<0.05, Table 2, Fig.2). The morphometrical parameters in control, AL, and MET groups were almos t identical.  Figure 3 shows the changes in MDA level and activities of antioxidant enzymes in tes tis tissues of different groups. There was a significant increase in MDA level along with a significant reduction of SOD and GPx activities in CIS-treated rats compared to the control group (P<0.05, Fig.3). Nevertheless, adminis tration of AL or MET together with CIS significantly reduced the MDA level and elevated antioxidant enzymes activities in comparison to the CIS group (P<0.05). There was no significant difference in MDA level and antioxidant enzymes activities in the AL and MET groups compared to the control group (Fig.3).

Comparison of serum tes tos terone level
As showed in Figure 3, tes tos terone level was significantly lower in CIS group rats compared to the other groups (P<0.05, Fig.3). Treatment of CIS rats by AL and MET significantly ameliorated the reduction of tes tosterone level (P<0.05). AL and MET groups presented no significant difference in the serum tes tos terone level compared to the control group (P>0.05, Fig.3).

Comparison of sperm parameters
A comparison of the groups with regard to sperm parameters is presented in Table 3. The sperm concentration, viability, and progressive and non-progressive motility decreased significantly in the CIS group compared to the control group but the percentage of abnormal sperm morphology increased (P<0.05, Table 3). By contras t, adminis tration of AL gel along with CIS could significantly improve the sperm parameters compared to the CIS group (P<0.05, Table 3). Also, treatment of CIS rats by MET significantly increased sperm concentration, viability and progressive motility and reduced abnormal morphology of sperm compared to the CIS group (P<0.05). Non-progressive motility increased in the CIS-MET group, but this change was not significant compared to the CIS group (Table 3).
No significant differences were observed in the sperm parameters between the AL and MET groups and the control group (Table 3).

Discussion
CIS-based chemotherapy induces gonadal toxicity and infertility by increasing oxidative s tress (5,6). Hence, adminis tration of antioxidant agents may be a useful s trategy in reducing CIS toxicity and preserve the fertilization capacity of patients receiving CIS.
The results of the present s tudy showed that CIS decreased relative weight and dimensions of the tes tis, and reduced the germinal epithelium thickness, and the diameter of seminiferous tubules. Additionally, his topathological changes such as tes ticular atrophy, desquamation, vacuolation of germinal epithelium, and reduction of spermatogenesis activity were observed in CIS-treated rats.
Loss of tes ticular weight and dimension in CIS-treated rats could be due to the inhibition of spermatogenesis, atrophy of tes ticular tubules, reduction of spermatogenic cells, and other degenerative alterations caused by CIS (25). These his tological damages may be explained by disruptions of the redox balance induced by CIS which result in DNA damage, lipid peroxidation, and inhibition of protein synthesis (4). Tes tis tissue is highly vulnerable to oxidative s tress because it has a high metabolic activity and considerable amount of highly unsaturated fatty acids (26). Free radicals impair different parts of the tes tis especially tes ticular germinal cells and lead to atrophy in tes ticular tubules and reduction of sperm generation (20,26,27).
Data from the present s tudy likely showed that CIS treatment impairs oxidant-antioxidant balance in tes ticular tissue so that it increased the levels of MDA and decreased antioxidant enzymes (SOD and GPx) activities, these results are in agreement with previous reports (27,28). The peroxidation of lipids is one of the toxic effects of CIS in the tes tis and MDA is produced as the end-product of this process; thus, MDA content is the bes t marker for measuring oxidative s tress and lipid peroxidation indirectly. Also, the increase in the MDA level may be related to DNA fragmentation as reported previously (29). The reductions of the antioxidant enzymes activities observed in this s tudy, are probably due to either direct effects of CIS on these enzymes or enhanced consumption of antioxidant enzymes for detoxifying free radicals generated by CIS (30).
We found a CIS-mediated decrease in serum tes tos terone concentration which is fundamentally consis tent with previous s tudies (27,29). Saral et al. (27) reported that the reduction of tes tos terone level induced by CIS results from a decrease in the number of Leydig cells or their dysfunction. Another hypothesis is that CIS inhibits tes tos terone synthesis by depressing the cytochrome P-450-dependent 17-α-hydroxylase level and decreasing the numbers of luteinizing hormone (LH) receptors in Leydig cells.
CIS treatment reduced sperm concentration, motility and viability and increased abnormal sperm morphology, consis tent with many reports that have indicated the side effects of CIS on sperm function (20,28). The alteration in sperm parameters of the CIS group was probably caused by prolonged exposure of the tes tis to CISinduced free radicals (20). Free radicals decrease the mitochondrial membrane potential in sperm cells which is associated with a decrease in adenosine triphosphate (ATP) production and inhibition of sperm motility (31). In addition, damage of the sperm cell membrane by CISinduced free radicals may be the cause for the decrease in sperm viability and motility and the increase in the morphological defects (32).
Erfani Majd et al.  In the present s tudy, adminis tration of AL gel at a dose of 400 mg/kg effectively inhibited the CIS-induced tes ticular oxidative s tress by decreasing the MDA levels and increasing the antioxidant enzymes activities. Also, our results clearly showed that AL treatment attenuated adverse effects of CIS on relative tes ticular weight and dimension, sperm parameters, tes tos terone level, and his tological changes of the tes tis.
These chemoprotective effects of AL a gains t CISinduced toxicity may be related to the antioxidant effect of AL, as reported in previous s tudies (33,34). Imaga et al. (33) reported that AL gel improves CISinduced oxidative damages in the kidney and liver of experimental animals. Also, Chatterjee et al. (34) indicated that adminis tration of AL along with CIS was associated with amelioration of antioxidant defense sys tem and diminution of CIS-induced nephrotoxicity. AL and especially its gel are highly spermatogenic and enhance male fertility by elevating sperm quality (10,11). AL increases spermatogenesis process via affecting spermatogenic cells and s timulating cell division, and increases tes tos terone hormone by s timulating Leydig cells (10,11,35).
Es takhr and Javdan (10) reported that AL significantly increased tes ticular weight, tes tos terone hormone, and sperm concentration and motility and decreased sperm abnormalities. Also, AL increases cAMP responsive element modulator (CREM) gene expression that has a key role in the regulation of the expression of genes that control spermatogenesis (11).
AL contains a large number of antioxidant compounds including vitamins (A, C, B, E), flavonoids, phenolic compounds, and polysaccharides (7). Vitamin E has the highes t antioxidant activity and plays a key role in the protection of plasma membrane agains t peroxidation by free radicals. Also, vitamin E improves tes ticular weight, germinal epithelium thickness, and diameter size of seminiferous tubule (36). Vitamin C in AL gel performs an important role in the integrity and fertility of semen and makes up to 65% of the total antioxidant capacity of seminal plasma. Also, vitamin C inhibits sperm agglutination and increases tes tos terone concentration (37). Furthermore, phenolic compounds and polysaccharides of AL have antioxidant capacity and prevent diseases induced by oxidative s tress (38). Therefore, because of its antioxidant properties, AL can reduce CIS-induced oxidative damages in tes tis tissue and can support spermatogenesis and protect spermatozoa agains t free radicals.

Conclusion
Our findings demons trated that oxidative s tress can play a significant role in the pathogenesis of CISinduced tes ticular and sperm injuries. Also, biochemical, hormonal, and his tological results sugges t that AL gel could be effective for prevention of gonadal toxicity induced by CIS in male Wis tar rats. This s tudy concluded that AL gel due to its potent antioxidant effect, can protect the tes ticular tissue from toxic damages caused by CIS.